Establishment of a Genetic Transformation System in Guanophilic Fungus Amphichorda guana

Min Liang,Wei Li,Landa Qi,Lei Cai,Wen-Bing Yin,Guocan Chen

doi:10.3390/jof7020138

Abstract

Fungi from unique environments exhibit special physiological characters and plenty of bioactive natural products. However, the recalcitrant genetics or poor transformation efficiencies prevent scientists from systematically studying molecular biological mechanisms and exploiting their metabolites. In this study, we targeted a guanophilic fungus Amphichorda guana LC5815 and developed a genetic transformation system. We firstly established an efficient protoplast preparing method by conditional optimization of sporulation and protoplast regeneration. The regeneration rate of the protoplast is up to about 34.6% with 0.8 M sucrose as the osmotic pressure stabilizer. To develop the genetic transformation, we used the polyethylene glycol-mediated protoplast transformation, and the testing gene AG04914 encoding a major facilitator superfamily transporter was deleted in strain LC5815, which proves the feasibility of this genetic manipulation system. Furthermore, a uridine/uracil auxotrophic strain was created by using a positive screening protocol with 5-fluoroorotic acid as a selective reagent. Finally, the genetic transformation system was successfully established in the guanophilic fungus strain LC5815, which lays the foundation for the molecular genetics research and will facilitate the exploitation of bioactive secondary metabolites in fungi.

Highlights

IntroductionSeveral reports have shown that endophytes are able to biosynthesize medicinally important phytochemicals and many novel bioactive natural products have been discovered in marine fungi [4,5]
Fungi growing in unique environments, such as endophytic fungi, marine fungi, and coprophilous fungi, often have special physiological and metabolic characteristics [1,2,3].Several reports have shown that endophytes are able to biosynthesize medicinally important phytochemicals and many novel bioactive natural products have been discovered in marine fungi [4,5]
Strain LC5815 and its transformants were routinely maintained on potato dextrose agar (PDA) or in potato dextrose broth (PDB) at 28 ◦ C in the presence of appropriate antibiotics (Table 1)

Summary

Introduction

Several reports have shown that endophytes are able to biosynthesize medicinally important phytochemicals and many novel bioactive natural products have been discovered in marine fungi [4,5]. Taxol (from the endophytic fungus Taxomyces andreanae) and destruxins (from marine fungus Beauveria felina) are examples of fungi-derived compounds that are widely used in the pharmaceutical and agrochemical industries [6,7]. Coprophilous fungi are rich sources of natural products given the inherent microbial competition in short-lived animal excrement [3]. They have received greater attention in recent years because of their ubiquity and ease of study [8]. G1-a14 [10], benzophenone and fimetarone derivatives from Delitschia confertaspora [11], and indole alkaloids from Aphanoascus fulvescens [12]

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